Specific protein-protein interactions are essential to nearly every biological process. Defining how specific interactions are made is critical for a full understanding of the biological system governed by the interacting proteins. Yeast two-hybrid analysis is a routine and powerful assay for examining protein-protein interactions that has been adapted to high-throughput screening (Fields, S. & Song, O. K., 340 NATURE 245-46 (1989), Chien et al., 88 PROC. NATL. ACAD. SCI. U.S.A. 9578-82 (1991)). In fact, large-scale yeast two-hybrid screening has been utilized to collect much of the binary protein interaction data available to date (Uetz et al., 403 NATURE 623-27 (2000), Ito et al., 98 PROC. NATL. ACAD. SCI. U.S.A. 4569-74 (2001), Stelzl et al., 122 CELL 957-68 (2005), Rual et al., 437 NATURE 1173-78 (2005), Walhout et al., 17 YEAST 88-94 (2000)). However, such large-scale yeast two-hybrid screens face numerous challenges, including strategies for cloning numerous genes into yeast two-hybrid vectors for expressing DNA-binding domain (“bait”) and activation domain (“prey”) fusions in yeast. Conventional techniques involving individual restriction digests and ligations for cloning genes into appropriate expression vectors can be impractical and expensive when dealing with thousands of genes. Several alternative systems for cloning yeast two-hybrid constructs based on DNA homologous recombination reactions have been described to date (Uetz et al., 2000, Walhout et al., 2000). Even though these techniques eliminate the need for restriction enzymes and ligases, they still require thousands of yeast transformations to generate a library of activation-domain fusion constructs (Uetz et al., 2000, Walhout et al., 2000).
The development of mating-based screens has facilitated the conversion of yeast two-hybrid from a directed, small-scale assay to a high-throughput one (Gyuris et al., 75 CELL 791-803 (1993), Finley, R. L. & Brent, R., 91 PROC. NATL. ACAD. SCI. U.S.A. 12980-12984 (1994), Bendixen et al., 22 NUCLEIC ACIDS RES. 1778-79 (1994), Fromont-Racine et al., 17 YEAST 95-110 (2000)). Mating-based screens rely on haploid yeast having two mating types, MATa and MATα, which fuse to form diploids (Herskowitz et al., 57 MICROBIOL. REV. 536 (1988)). In these assays, DNA-binding and activation-tagged proteins are expressed in different haploid strains and are brought together through mating (Gyuris et al., 1993). Large numbers of individual protein-protein interactions can then be tested (Gyuris et al., 1993, Finley and Brent, 1994, Fromont-Racine et al., 1997, Walhout A. J. M. & Vidal, M., 24 METHODS 297-306 (2001)). However, establishing efficient strategies to mate large sets of bait and prey yeast strains to sample all possible combinations of interactions has also proven difficult (Uetz et al., 2000, Parrish et al., 17(4) CURR. OPIN. BIOTECHNOL. 387-93 (2006)).
Another limitation of the classic yeast two-hybrid system is simultaneous detection of multiple protein-protein interactions. Ternary-protein complexes in which a protein requires interaction with a second protein in order to bind a third protein, and where a third protein only binds to a composite site formed by the association of the first and second proteins, can be analyzed using a yeast “three-hybrid system” (Zhang, Y. & Chan, D. C., 104 PROC. NATL. ACAD. SCI. U.S.A. 18526-18530 (2007)). However, assays for simultaneous screening of multiple interacting partners have yet to be described, and current technology requires multiple rounds of screening (Fromont-Racine et al., 1997).
Yeast two-hybrid assays have also been useful in mapping binary protein-protein interfaces (Lehming et al., 92 PROC. NATL. ACAD. SCI. U.S.A. 92, 10242-10246 (1995), Steffan et al., 18 MOL. CELL. BIOL. 3752-61 (1998), Vidal et al., 93 PROC. NATL. ACAD. SCI. U.S.A. 93, 10315-10320 (1996)). These assays often utilize PCR-based random mutagenesis followed by homologous recombination and gapped plasmid repair to construct a library of mutated proteins to screen for disruption of interaction with its binding partner (Lehming et al., 1995, Steffan et al., 1998). However, a common issue with this type of approach is the generation of uninformative mutations, such as truncations, frameshifts, or any mutations that affect the stability, processing or folding of the protein. To eliminate isolation of these trivial results, a “reverse yeast two-hybrid” system was developed (Vidal et al., 93 PROC. NATL. ACAD. SCI. U.S.A. 93, 10321-10326 (1996)) to include a two-step selection process. The first step is a negative selection for mutations that impair a protein-protein interaction and the second step is a positive selection for a subset of those mutations that maintain expression of full-length stable proteins (Vidal et al., 93 PROC. NATL. ACAD. SCI. U.S.A. 93, 10321-10326 (1996)). While this two-step selection accomplishes both identification of disruptive mutations and elimination of trivial mutations, it can only be used to study the interactions of two proteins partners.
To date, mutagenesis-based yeast two-hybrid screens have only been utilized to examine the interactions of binary protein-protein interactions, and have not been extended to the analysis of multiple interacting partners.